Abstract:
The delayed transition to reproductive maturity in horticulturally important crops substantially affects their breeding and large scale cultivation. Being woody perennials, the long juvenile phase also impedes the cultivation of Actinidia spp. including kiwifruit and kiwiberry plants. Manipulation of genetic regulators of flowering has the capacity to develop early flowering plants, which can further give rise to improved cultivars with important agronomic traits. Previous studies have characterised several genetic regulators of flowering in A. chinensis, but is centrally regulated by antagonistic expression of FT-like and CEN-like genes. Among these, manipulation of flowering repressor CEN-like genes, also in A. chinensis, have successfully altered plant traits to give favourable agronomic traits like early flowering, determinate meristems and compact size.
This study focuses on targeting CEN4 in A. arguta plants to alter and analyse its role in regulating flowering time and plant architecture. One approach was to develop plant lines expressing a transactivation domain VP16, downstream of AcCEN4 using Agrobacterium-mediated plant transformation. Similar construct expressed in Arabidopsis showed that VP16 converts the flowering repressor to an activator and could also be inherited dominantly. Moreover, genetic dominance can allow the development of specific traits for breeding in tetraploid crops, which cannot be achieved by recessive genotypes generated by mutagenesis of genes. Although, both A. arguta and A. chinensis plants lines constitutively expressing AcCEN4-VP16 did not transition to reproductive maturity in the observed period of 2-3 months after acclimatisation.
Meanwhile, this study also genotyped previously generated AaCEN4-edited A. arguta plants to detect mutations or indels rendered by a CRISPR/Cas9 PTG construct. Being tetraploids with unknown allelic information of AaCEN4, wildtype plants were first genotyped which identified 24 polymorphisms defining each allele. Wildtype genotyping was also important for distinguishing between allelic polymorphisms and mutations/indels occurring from gene editing. This information was then used to identify alleles in AaCEN4-edited lines, followed by analyses of guide RNA targeted regions. Although, no mutations or indels were observed in the targeted regions of AaCEN4 and was attributed to inefficient guide design.
Overall, the study explores different approaches of genetic manipulation of AaCEN4 in A. arguta plants in order to generate early flowering lines and provides insights on improving their efficiency.